Scleroderma. Chronic multisystemic disease characterized by vasculopathy, variable degree of inflammation, and fibrosis

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1 Scleroderma Chronic multisystemic disease characterized by vasculopathy, variable degree of inflammation, and fibrosis Incidence cases/million Female:male 5:1 Pulmonary fibrosis common, severe in 16%. Pulmonary hypertension occurs in 50% of cases and can lead to cor pulmonale. Pulmonary complications are the leading cause of death in this disease. Clinical Features of Scleroderma (Systemic sclerosis; SSc) Typical facial features in advanced SSc Digital ulceration from vascular damage 1

2 CREST: Calcinosis, Raynaud s phenomenon, Sclerodactyly, Esophageal dysmotility, Telangiectasias Telangiectasias Ranaud s phenomenon Calcinosis Sclerodactyly Organ Involvement in Scleroderma Differential organ involvement in SSc. The earliest pathological feature of SSc in the skin is vasculopathy with endothelial cell activation. Later, inflammation develops and finally fibrosis is prominent. Similar processes are likely to occur in all lesional tissues, leading to organ dysfunction. Cardiac, renal, lung and gut complications are the main causes of SSc-related mortality. From: Denton and Black Trends Immunol. 26:596,

3 Pulmonary Manifestations of Scleroderma Normal Pulmonary Pulmonary Hypertension Fibrosis Major Clinical Sub-types of Scleroderma Limited cutaneous SSc (60% of cases) No skin sclerosis proximal to knees or elbows. Longstanding Raynaud's phenomenon is typical. Most commonly associated autoantibody is anti-centromere Ab (ACA). A subgroup of these patients has manifestations of CREST (calcinosis, Raynaud's, esophageal dysmotility, telangiectasis). Isolated pulmonary hypertension and gastrointestinal tract dysmotility are the most common severe manifestations. Lung fibrosis, renal and cardiac involvement occur less often than in dcssc. 3

4 Major Clinical Sub-types of Scleroderma Diffuse cutaneous SSc (30% of cases) The cardinal feature is skin sclerosis proximal to the knees and elbows. Raynaud's phenomenon is universal but might manifest simultaneously or shortly after the development of skin sclerosis. Inflammatory features are prominent during the first 3 years of disease. Skin involvement often diminishes within two years. ACA is rarely present, anti-scl-70 (topoisomease-1) or anti-rna polymerase is typical. There is a high frequency of interstitial lung disease, renal crisis and cardiac involvement. Major Clinical Sub-types of Scleroderma Scleroderma overlap syndromes Their features include those of limited or diffuse cutaneous SSc, together with those of one or more additional autoimmune rheumatic diseases, such as polyarthritis, myositis or SLE. Often associated with anti U1-ribonucleoprotein (U1-RNP), U3-RNP or polymyositis-scleroderma (PM-Scl) autoantibody reactivity. Localized scleroderma Morphea scleroderma causes patches of hard skin that can persist for years. Linear scleroderma causes bands of hard skin across the face or an extremity, rarely involving muscle or bone Typically carries a good prognosis 4

5 Genetics of Scleroderma Family history associated with increased risk of developing disease, but risk is only 1% for any individual. Concordance for both mono- and dizygotic twins is 5%. However, gene expression profiling of cultured dermal fibroblasts from monozygotic twins of an index case show a similar pro-fibrotic signature 46% of the time. Genetic studies indicate an association of scleroderma with polymorphisms in the promoters of the TNF, MCP-1, and CD19 genes. Although genetic studies have suggested an association of the HLA- DRB1*01 allele with anti-centromere antibodies, association of ACA with polymorphisms in the TNF promoter was even stronger, suggesting linkage disequilibrium. The Role of B Cells in Scleroderma In the blood of patients with scleroderma, naive B cells are increased in number, while memory B cells and plasmablasts/early plasma cells are diminished. Memory B cells express higher levels of CD80 and CD86, and thus are chronically activated, possibly due to CD19 over-expression. CD95 expression is also increased on memory B cells, leading to augmented CD95-mediated apoptosis. The continuous loss of memory B cells and plasmablasts/early plasma cells may increase naive B-cell production in bone marrow to maintain the B-cell homeostasis in systemic sclerosis. From: Fujimoto and Sato: Curr. Opin. Rheumatol. 17:746,

6 The Role of B Cells in Scleroderma Legend From: Fujimoto and Sato: Curr. Opin. Rheumatol. 17:746, 2005 Do B Cells and Autoantibodies Play a Causative Role in Scleroderma? 6

7 Autoantibodies to the PDGF Receptor Stimulates Production of Reactive Oxidants Oxidant production triggered by sera-derived IgG upon incubation with fibroblasts over-expressing the PDGF receptor. N, normal controls; SSc, scleroderma; PRP, primary Raynaud s phenomenon ; SLE, systemic lupus erythematosis; RA, rheumatoid arthritis; ILD, interstitial lung disease; AG 1296, PDGFR kinase inhibitor. From: Baroni et al., New Engl. J. Med. 354:2667,

8 Scleroderma-derived Antibodies to the PDGF Receptor Stimulate Cell Signaling and Collagen Production IP: PDGF receptor Blot Legend: FCS, fetal calf serum; AG1478, EGFR tyrosine kinase inhibitor; AG1296, PDGFR kinase inhibitor Adapted from: Baroni et al., New Engl. J. Med. 354:2667, 2006 Auto-antibodies to the PDGF Receptor: The Pathogenesis of Scleroderma Revealed? Unlike normal fibroblasts, fibroblasts in scleroderma increase the expression of PDGFR in response to TGF-, rendering the cells more sensitive to PDGF. The Ras ERK1/2 ROS signaling pathway is triggered by PDGF or anti- PDGFR, which then activates NADPH oxidase (NOX1) to produce reactive oxygen species (ROS). These, in turn, activate extracellular signal-regulated kinases 1 and 2 (ERK1/2), which induce the H-ras gene. This signaling loop is present in normal fibroblasts but is relatively amplified in fibroblasts in patients with scleroderma. From: Tan, New Engl. J. Med. 354:2709,

9 Potential Therapeutics for Scleroderma Based on Insight Into its Pathogenesis From: Baroni et al., New Engl. J. Med. 354:2667, If chronic over-stimulation of the PDGFR is required for the development and/or progression of scleroderma, then inhibition of PDGFR kinase activity (AG 1296), Ras post-translational processing (FTI 277), or ERK MAP kinase (PD 98059) may prove therapeutic. Therapeutic Approach to the Treatment of Scleroderma From: Denton and Black Trends Immunol. 26:596,

10 Fibrosis Can Occur in Any Organ and Can Lead to Irreversible Organ Damage Various theories have been proposed for the pathogenesis of fibrosing diseases. One view is that fibrosis represents a pathological variant of wound healing. An alternate view is that fibrosis is due to unresolved inflammation. Another view is that fibrosis results from an imbalance in the activities of proteases and anti-proteases. Regardless of the precise etiology of fibrosis, the pathological deposition of collagen and other components of the extracellular matrix results from persistence of mesenchymal cells assuming a fibroblastic or myofibroblastic phenotype, producing copious amounts of components of the ECM. Examples of Fibrosis Normal glomerulus Hepatitis C cirrhosis Glomerulosclerosis Retroperioneal fibrosis 10

11 Idiopathic Pulmonary Fibrosis (IPF) Normal IPF From: Best et al., Radiology 228:407, 2003; Wittram et al. Radiographics 23:1057, Three questions worth pondering 1. What cells and molecules participate in fibrosis? 2. What is the pathogenesis of fibrosis? 3. Can we intervene therapeutically and retard or reverse fibrosis? 11

12 Fibrosis Results from the Inappropriate Deposition of Extracellular Matrix Collagen Fibronectin Laminin Proteoglycan Integrin Two Engaging Members of the ECM Fibronectin Laminin 12

13 Diversity in Cells and Component of the ECM Collagen Anchor Proteoglycan Cell-Surface Receptor Cells I fibronectin chondroitin and dermatan sulfates integrin fibroblasts II fibronectin chondroitin sulfate integrin chondrocytes III fibronectin quiescent hepatocytes, heparan sulfate and integrin epithelial; assoc. heparin fibroblasts IV laminin heparan sulfate and heparin laminin receptors all epithelial cells, endothelial cells, regenerating hepatocytes V fibronectin heparan sulfate and heparin integrin quiescent fibroblasts VI fibronectin heparan sulfate Iitegrin quiescent fibroblasts Do not memorize this list Fibrosis is triggered by growth factors, cytokines, and peptides 13

14 Important Pro-fibrotic Growth Factors Growth factors TGF-, PDGF, FGF-2 (basic FGF), IGF-I, Connective tissue growth factor (CTGF), EGF Cytokines Th2 cytokines (IL-4, IL-13); IL-1, IL-6 Peptides Endothelin I, Ang-II Chemokines C-C chemokines Do memorize this list Activation of Latent TGF- : Not So Simple TGF- is synthesized by virtually all types of cells, in the form of an inactive homodimeric propeptide, pro-tgf-. After TGF- is cleaved from the pro-peptide, additional downstream events control the extent to which TGF- is available. The small latent complex, composed of TGF- and its latency-associated peptide, and the large latent complex, composed of TGF-, latency-associated peptide, and the latent TGF- binding protein control where and when active TGF- is made available. The matrix protein thrombospondin-1 binds to the latencyassociated peptide, which results in the activation of TGF-, an essential step for the binding of TGF- to its receptors. Plasmin, some MMPs, and two members of the integrin family also activate the latent complexes of TGF-. From: Ugust and Suthanthiran New Engl. J. Med. 354: 2721,

15 Basic Paradigm of TGF- Signal Transduction Legend From: Yokote et al., Trends Cardiovasc. Med. 16:240, 2006 Anti-proliferative and Pro-fibrotic Properties of TGF- Signaling Revealed by Gene Targeting in Mice Enhanced neointimal hyperplasia and reduced matrix deposition in the arteries of Smad3-null mice upon injury. Photomicrographs showing representative cross sections of hematoxylin/eosin-stained (A and B) and Masson's trichrome-stained (C and D) femoral arteries from wild-type (A and C) and Smad3-null (B and D) mice 3 weeks after endothelial injury by photochemically induced thrombosis method. Arrows indicate the positions of the internal elastic lamina. L, vascular lumen. Frrom: Circ Res 96:904,

16 The Myofibroblast, a Collagen-producing Contractile Cell Involved in Wound Healing -smooth muscle actin The Hepatic Stellate Cell is the Myofibroblast-equivalent in Cirrhosis From Bataller and Brenner, J. Clin. Invest. 115:209,

17 What are the cellular origins of fibroblasts and myofibroblasts? Local proliferation of mesenchymal cells Influx of blood-borne fibroblast precursors (often termed fibrocytes ) Epithelial-mesenchymal transformation (EMT) Use of Bone Marrow Chimeras to Determine the Cellular Origin of Fibroblasts Representative lung sections from bleomycin (BLM)-treated (a c) or saline-treated (d f) GFP BM chimera mice were evaluated at day 28 after BLM or saline treatment. The H&E-stained sections revealed severe distortion of lung alveolar architecture due to extensive fibrosis (a and b, at x40 and x200, respectively). (c) Numerous GFP + cells in densely cellular fibrotic lesions were easily identifiable by fluorescence microscopy in BLM-treated lungs. x200. In contrast, H&E-stained lung sections from saline-treated GFP BM chimera mice showed normal lung architecture (d and e, at x40 and x200, respectively), with a few scattered GFP + cells visualized by fluorescence microscopy (f, at x200). Insets in c and f showed the light-microscopic appearance of the respective sections examined by fluorescence microscopy. x200. From: Hashimoto et al., J. Clin. Invest. 113:243,

18 GFP+ Col I+ Cells Derived from the Bone Marrow Populate Lungs During Experimental Pulmonary Fibrosis in Mice Representative flow cytometry tracings of cells isolated from lungs of bleomycin- and control saline-treated mice and stained with antibodies that detect Col I. Insets show staining of cells using an isotype-matched control antibody. From: Hashimoto et al., J. Clin. Invest. 113:243, Epithelial-mesenchymal Transformation (EMT) (a) Schematic of EMT. Epithelial cells (in blue) adhere to each other through adherens junctions, using E-cadherin (E-cad), and their desmosomes (tight junctions) constituted by various proteins such as desmoplakin (dp). Mesenchymal cells (in red) are neither adherent nor apically polarized and have low expression of E-cadherin and desmoplakin. The intermediate filament protein vimentin (vim) is induced in mesenchymal cells. (b g) EMT in a human squamous cell carcinoma line. Prior to EMT (b) and after EMT (c). E-cadherin (red in d and e) and vimentin (f and g) immunolocalization in epithelial (d and f) and mesenchymal cells following EMT (e and g). Adapted from Larue and Bellacosa, Oncogene 24:7443,

19 E-Cadherin Appears to be a Critical Regulator of EMT Is repression/disruption of E-cadherin expression sufficient to induce EMT? The Canonical Wnt Pathway Leads to Activation of a Subset of Genes Important in Development N.B.: Do not memorize 19

20 Repressing E-Cadherin Triggers EMT TGF- 2/3 Wnt P cytoplasm Smad2 Smad4 TCF TCF -cat nucleus e-cadherin promoter e-cadherin Twist Slug Snail N.B.: Do not memorize FGF, TGF-? IL-4, IL-13 Summary 1. Scleroderma is an autoimmune disease involving multiple organs. It is characterized by a vasculopathy, varying degrees of inflammation, and fibrosis. Several major clinical variants of scleroderma have been described. 2. Pulmonary complications of scleroderma are common and severe in substantial minority. Death is due to severe pulmonary hypertension, pulmonary fibrosis, or both, ultimately leading to cor pulmonale. 3. Autoantibodies play an important role in the pathogenesis to scleroderma. Agonistic autoantibodies against the PDGFR have been isolated from patients with scleroderma. 4. Various theories of fibrogenesis have been proposed. Regardless of the precise etiology, fibrosis is due to the excessive deposition of ECM by the major collagenproducing cells in the body, fibroblasts and myofibroblasts. 5. Fibroblasts arise from a combination of local proliferation of resident mesenchymal cells, EMT, and influx of fibroblast precursors from the bone marrow. 6. Among the various pro-fibrogenic growth factors, TGF- usually plays an important role in fibrosis. TGF- triggers increased production of ECM proteins, induces EMT, and typically induces proliferation (rather than cell cycle arrest) in fibroblasts. 20